Project Summary Cell polarization is of fundamental importance to the morphogenesis of living organisms. Polarized epithelial cells comprise the foundation for the majority of organs in the mammalian body, and defects in polarity and intercellular junctions are responsible for multiple diseases of the kidney, skin, and intestine. A deep knowledge of epithelial morphogenesis is essential, therefore, to understanding the development both of normal tissues and of many human diseases. The PAR proteins are key components of the polarity machinery and interact with other polarity proteins, such as CRUMBS, PALS1, and atypical protein kinase c (aPKC). Some of these factors play important roles beyond those associated with polarity, including cell division and survival. In previous funding periods we made substantial contributions to the current understanding of cell polarity complexes and their functions. Recently, several interactions with membrane transport factors have been uncovered, illuminating a long-suspected link to polarized vesicle transport. Nonetheless, the molecular mechanisms coupling polarity proteins to vesicle traffic are still not well understood. In this project, we will exploit new technologies in gene-editing and microscopy to investigate with unprecedented resolution vesicle tethering at the plasma membrane and its regulation by PAR polarity proteins, apical membrane formation, and novel factors involved in this process. Using CAS9 gene-editing, we have created multiple epithelial cell lines that express functional, fluorophore-tagged alleles of the endogenous exocyst subunits, exocyst regulators and multiple polarity proteins, using both mouse mammary epithelial cells and human induced pluripotent stem cells (hIPSCs). Analysis of these lines by multi-channel total internal reflection fluorescence microscopy (TIRFM) or near-TIRF (HILO) imaging, and quantitative mass spectrometry (MS) will enable the resolution of long-standing questions about the regulation of exocyst and polarity complex dynamics. Because CRB regulates apical size and is a key regulator of HIPPO signaling, its concentration at the apical membrane must be closely regulated, but the mechanisms for this control are not understood. We developed a new assay to study apical protein dynamics, using a fusion of GFP-binding peptide (GBP) to the extracellular domain of CRB3. Addition of recombinant GFP rapidly tags this protein. We exploited this system to perform a genome-wide CRISPR knockout screen to identify factors that regulate apical CRB3 localization. Candidate proteins, and known factors such as the polarity proteins PAR6 and aPKC will be functionally analyzed using HILO imaging and a system to synchronously release CRB3 after trapping it in the ER. Together these studies will provide important new insights into the connections between vesicle traffic and the cell polarity machinery.